Insights into mobile genetic elements and the role of conjugative plasmid in transferring aminoglycoside resistance in extensively drug-resistant Acinetobacter baumannii AB329

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (blaOXA-51, blaADC-25, blaOXA-23, blaTEM-1D), aminoglycoside resistance (aph(3′)-Ia, aph(3″)-Ib, aph(6)-Id, armA), tetracycline resistance (tet(B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(3′)-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs.


INTRODUCTION
Acinetobacter baumannii is a bacterium that is a major cause of nosocomial infection, especially in intensive care units (ICUs). In recent decades, the prevalence of extensively drug-resistant A. baumannii (XDRAB) has been rapidly increasing worldwide. Numerous antibiotic resistance genes (ARGs) have been detected in the genomes and mobile genetic elements (MGEs) of XDRAB, and they have been found to be responsible for the spread of antibiotic resistance. A variety of MGEs have been described in A. baumannii. In silico analysis detected various ARGs located in A. baumannii conjugative plasmids (Makke et al., 2020;Martins-Sorenson et al., 2020). A conjugative plasmid is a self-replicating plasmid that generally carries all the genes required for bacterial conjugation. The conjugative plasmid requires the origin of transfer and the tra operon, which is important for generating the F-pilus that is needed for transferring genetic materials. Plasmid classification of A. baumannii can be divided into 23 different groups (GR1-GR23) based on the nucleotide identity of the replicase genes (repA) using the polymerase chain reaction-based replicon typing (AB-PBRT) method (Bertini et al., 2010;Salgado-Camargo et al., 2020). Kongthai et al. (2021) reported the detection of tra genes in XDRAB strains harbored in plasmid GR6. This GR6 plasmid is responsible for the dissemination of drug resistance genes such as bla  and aphA6 (Saranathan et al., 2014;Leungtongkam et al., 2018a). Additionally, A. baumannii plasmids were also classified into 21 lineages (LN_1-LN_21) based on their core DNA sequence backbones and plasmid incompatibility groups (Salgado-Camargo et al., 2020).
The two major clones responsible for A. baumannii outbreaks worldwide are global clone 1 (GC1) and global clone 2 (GC2) but are referred to as international clones 1 (IC1) and 2 (IC2) (Hamidian & Nigro, 2019). The global distribution of CRAB has been heavily influenced by the spread of IC2 isolates, and most of these clones are defined as sequence type 2 (ST2) according to the Institut Pasteur MLST scheme (Hamidian & Nigro, 2019). IC2 is the predominant clonal lineage in Spain, South Korea, China, Australia, Singapore, and Thailand (Hamidian & Nigro, 2019, Khuntayaporn et al., 2021. Our previous study of 339 A. baumannii isolates collected from four hospitals in Thailand revealed 7.9% XDRAB among the total isolates collected (Leungtongkam et al., 2018a). We found an outbreak clone of XDRAB in one hospital with the same ST type and plasmid group that belonged to IC2 (Kongthai et al., 2021). All of them were ST98 (Pasteur) and contained GR2 and GR6 plasmids. Little is known about the role of conjugative plasmids in the functioning of XDRAB. The use of whole-genome sequencing (WGS) technology can assist in tackling antimicrobial resistance, virulence determinants, and MGEs in A. baumannii (Makke et al., 2020;Martins-Sorenson et al., 2020). Combined with short-and long-read sequencing, WGS will be able to resolve an accurate and complete genome and plasmid structure. Thus, this study aimed to obtain the complete genome sequence of XDRAB and to characterize the MGEs and the role of the plasmid in transferring ARGs.

Bacterial strains and antibiotic susceptibility testing (AST)
A. baumannii AB329, which is a phage-susceptible XDRAB strain, was isolated from patient sputum obtained from our previous study (Leungtongkam et al., 2018a). This strain is representative of the outbreak clone obtained from a hospital in east Thailand, and it was collected from November 2013 to February 2015 (Leungtongkam et al., 2018a). Antimicrobial susceptibility testing (AST) was performed as previously described (Kongthai et al., 2021). The AST results were interpreted according to the Clinical Laboratory Standard Institute (CLSI, 2020). The protocol was approved by the Naresuan University Institutional Biosafety Committee, and the project number was NUIBC MI 63-07-21.
Assay Kit in a Qubit R 2.0 Fluorometer (Life Technologies, CA, USA) prior to sequencing with both short-read (Illumina paired-end) and long-read (PacBio, Menlo Park, CA, USA) sequencing systems. For short-read sequencing, paired-end sequencing libraries (2× 250 bp) were constructed using the Nextera XT sample preparation kit following the manufacturer's suggestions, and they were sequenced using the Illumina MiSeq platform. The Illumina reads were trimmed with Sickle v1.33 using the default parameters (Joshi & Fass, 2011). For long-read sequencing, a large insert library (10 kb) was constructed and sequenced on the PacBio RS platform (Pacific Biosciences, Menlo Park, CA, USA). A hybrid assembly was conducted with the Illumina trimmed reads and PacBio reads using Unicycler v0.4.8.0 with the default settings (Wick et al., 2017). Unicycler automatically identified and trimmed the overlapping ends, and circular sequences were rotated to dnaA.

Genome annotation and bioinformatic analysis
The assembled circular chromosome and plasmids were functionally annotated using Prokka v1.12 with the default options. MLST types were determined in silico using the MLST database (https://pubmlst.org/organisms/acinetobacter-baumannii). The complete genomes of 292 A. baumannii strains retrieved from the NCBI database in December 2021 were used to identify the core genome (Table S1). A single-nucleotide polymorphism (SNPs) phylogenetic tree of the core genome was reconstructed using CSI Phylogeny with the default settings (Kaas et al., 2014). This tree was visualized and edited using Interactive Tree of Life (iTOL) (https://itol.embl.de/). A pangenomic analysis was executed using Roary v.3.13.0, which compared the five closest related genomes, including A. baumannii NIPH17_00019 (AP024415.1), A. baumannii XH856 (CP014541.1), A. baumannii KAB02 (CP017644.1), A. baumannii KAB06 (CP017652.1) and A. baumannii KAB05 (CP017650.1). Then, the output was illustrated using R studio as described in https://github.com/IamIamI/pADAP_project/tree/master/Roary_stats. The average nucleotide identity (ANI) was calculated using FastANI v.1.3 to estimate the wholegenome similarity among the two XDRAB strains, which were obtained from hospitalized patients in Thailand (Kongthai et al., 2021), and the five closest related genomes identified from the pan-genome analysis. Antimicrobial resistance and virulence genes were retrieved using the Comprehensive Antibiotic Resistance Database (CARD) and VFanalyzer (Liu et al., 2019), respectively. The large-scale BLAST score ratio (LS-BSR) pipeline was utilized to compare the virulence and drug resistance genes with 292 A. baumannii genomes (Table  S1). A BSR value of 0.4 and above was interpreted as the presence of genes, and a BSR value below 0.4 was inferred as gene absence (Sahl et al., 2014;Yakkala et al., 2019). Then, these BSR values were used to build a hierarchical clustering heatmap using the R packages pheatmap and tidyverse. MGEs were detected using MobileElementFinder (Johansson et al., 2021). The presence of prophage sequences in the genome of A. baumannii AB329 was analyzed using the PHAge Search Tool Enhanced Release (PHASTER) online server (Arndt et al., 2016). Prophage open reading frames (ORFs) were examined as described in a previous study (Fu et al., 2017). The prediction of genomic islands (GIs) and AbaR was conducted by running BLASTn. A set of nucleotide queries of GIs identified in A. baumannii ACICU was used for BLASTn searching (Di Nocera et al., 2011;Thummeepak et al., 2020).
Plasmid comparison and the identification of the AbaR structure were accomplished using Easyfig version 2.1 (Sullivan, Petty & Beatson, 2011). The complete genome was deposited in the NCBI GenBank database under the accession numbers CP091452 (chromosome), CP091453 (pAB329a), and CP091454 (pAB329b).

Conjugation experiment
A broth-mating conjugation assay was performed according to a previously published protocol with minor modifications (Leungtongkam et al., 2018b). Overnight cultures of the donor (A. baumannii AB329) and the sodium azide resistance recipient (A. baumannii NU13R) were adjusted in 0.85% NaCl until the cell suspensions reached a turbidity equal to a McFarland value of 0.5, which was measured using a densitometer (SiaBiosan, Riga, Latvia). Equal volumes (250 µl) of adjusted cell suspensions of the donor and the recipient were mixed in 500 µl of 2× Luria-Bertani (LB) broth and incubated for 4 h at 37 • C. Transconjugants were selected on three LB agar plates containing 250 µg/ml sodium azide (negative control), 20 µg/ml kanamycin (negative control), and 250 µg/ml sodium azide plus 20 µg/ml kanamycin.

Complete genome sequence of A. baumannii AB329
The complete genome sequence of A. baumannii AB329 generated by short-and long-read sequencing revealed a circular chromosome 3,948,038 bp in length with 39% GC content, and it contained two plasmids (pAB329a and pAB329b) ( Table 1). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. The Prokka prokaryotic genome annotation system identified 18 rRNAs, 72 tRNAs, 3,837 ORFs, and a total of 3,747 protein-coding genes on the main chromosome of AB329 (Table S3). Genes for tRNAs and rRNAs were detected only in the chromosome (Table 1). pAB329a contained 12 ORFs, while pAB329b contained 113 ORFs. AB329 was assigned to MLST type 1166/98 (Oxford/Pasteur) and was found to belong to the IC2 lineage.
We also compared the genome of A. baumannii AB329 with two XDRAB isolates from two different hospitals in Thailand as previously described (Kongthai et al., 2021). The ANI (%) values of A. baumannii AB329 with A. baumannii AB140 and A. baumannii AB053 were 99.72% and 99.27%, respectively (Table S4). A pangenome of A. baumannii AB329 consisting of 4,628 genes represented the core, shell, and cloud genomes (Fig. 1B). The core genome represented a pool of conserved genes that were present in all genomes and included 3,238 genes. The accessory genes, which included shell genes (genes present in two or more strains) and cloud genes (genes only found in a single strain), constituted a total of 1,390 genes.
ISAba125 was detected only in the plasmid, and it bracketed the aph (

Conjugative transfer of the aminoglycoside resistance gene of plasmid pAB329b
To investigate the role of pAB329b in the transfer of ARG, we performed a conjugation assay to study the plasmid's ability to transfer the aminoglycoside resistance gene to sodium azideresistant A. baumannii NU13R (recipient). As shown in Table 2, aminoglycoside resistance could be transferred from the donors (A. baumannii AB329) to the recipient (A. baumannii NU13R). The conjugation frequency was approximately 1.2× 10 −7 . The resistance genes, plasmid typing results, and antibiotic susceptibility of the donors, the recipient, and the transconjugants are shown in Table 2. The transconjugant MIC of kanamycin was > 64 µg/ml, which is approximately 32-fold higher than the MIC of kanamycin on the recipient strains. PCR amplification of aminoglycoside resistance genes (aph(3 )-VIa) and traU revealed their presence only in donors and transconjugants ( Table 2). The plasmid GR6 was detected in the transconjugant NU13R-pAB329b.

DISCUSSION
The incidence of XDRAB infection has increased, resulting in hospital outbreaks worldwide, including in Thailand. In this study, we investigated the genome features and MGEs of the XDRAB strain AB329. The complete genome sequence of A. baumannii AB329 was 3.9 Mb compared to that of previously reported XDRAB isolates, which ranged from 3.8 to 4.0 Mb (Chopjitt et al., 2020;Si-Tuan et al., 2020;Makke et al., 2020). The most dominant sequence type of multidrug-resistant A. baumannii (MDRAB) in Thailand was found to be ST2 (Pasteur), belonging to IC2 (Khuntayaporn et al., 2021;Chukamnerd et al., 2022), and the XDRAB ST types reported in Thailand were ST2, ST16, and ST1479 (Chopjitt et al., 2020;Kongthai et al., 2021). We found that the ST type of the A. baumannii AB329 strain was ST98 (Pasteur). ST98 is closely related to ST2 since only the cpn60 locus from seven loci of MLST typing is different. To date, the ST98 clone has been detected in the carbapenem-resistant A. baumannii (CRAB) strain isolated from Portugal (Silva et al., 2021). An analysis of the clonal relationship of A. baumannii AB329 among 292 A. baumannii isolates worldwide (Table S2) (Table S4). These bacterial strains might share a common ancestor since all six strains shared conserved homologous core genes (approximately 3,300 genes) (Harris et al., 2003). In addition, A. baumannii KAB02, KAB05, KAB06, AB329, and NIPH17  (Table S4). All ARGs were detected in the chromosome except the aph (3 )-VIa gene, which was found in pAB329b. These results implied that the AGRs can be rapidly transferred or passed from parent to offspring and can cause clone outbreaks in the hospital. To date, most A. baumannii strains display intrinsic resistance genes such as bla OXA-51 and bla ADC (bla AmpC ), while acquired resistance was detected in 19% to 31% of bacterial isolates (Kyriakidis et al., 2021). The antibiotic susceptibility pattern of A. baumannii AB329 revealed high resistance to many beta-lactam antibiotics (Table S2). We detected the intrinsic resistance genes bla OXA-51 and bla ADC-25 as well as bla OXA-23 and bla TEM−1D in the genome of A. baumannii AB329. However, other beta-lactamase genes that were previously reported, such as bla PER−1 , bla NDM−1 , bla SPM , bla SIM , bla VIM , bla GIM , and bla IMP , were not detected (Leungtongkam et al., 2018a;Hassan et al., 2021;Kongthai et al., 2021). In addition, four classes of efflux pumps, including the MFS, RND, SMR, and MATE families, are associated with the antimicrobial resistance of A. baumannii (Abdi et al., 2020). Consistent with a previous report, numerous virulence factors were detected in A. baumannii AB329 (Leal et al., 2020). Most of the virulence genes were detected in the other 292 A. baumannii strains isolated worldwide, and compared to the ARG patterns, the virulence gene patterns of XDRAB were not considerably different among the 292 A. baumannii strains ( Figs. 2A and  2B). These findings indicated that all A. baumannii strains were derived from the same ancestor and employed the same pathogenic mechanisms to cause disease. In contrast, horizontal gene transfer of ARGs is important for the difference in ARG patterns, which leads to a critical problem in the treatment of A. baumannii infection. Many of the virulence genes and ARGs are located in MGEs, such as plasmids, GIs, transposons (Tn), and prophages. These elements can move between genomes through bacterial HGT. In this study, we detected pAB329b, a novel, uncharacterized, 87-kb plasmid. The genome structure of pAB329b is a conjugative plasmid classified as the GR6 plasmid and belongs to LN_1 as well as pACICU2 (Salgado-Camargo et al., 2020). pAB329b might be derived from pACICU2 since it shares the same DNA backbone. A. baumannii strains carrying pACICU2 were isolated in 2005 in Italy, pAB329b was isolated in 2015 in Thailand, and pAB329b acquired the aph(3 )-VIa gene by horizontal gene transfer (Table  S5).
Conjugation experiments demonstrated that amikacin resistance could be transferred from A. baumannii AB329 donors to recipient, sodium azide-resistant A. baumannii isolates. We detected the aph (3 )-VIa gene, plasmid GR6 and traU gene from the tra operon, which is important for generating the F-pilus in transconjugants. A previous study determined that the bla OXA-23 , bla PER−1 , and aphA6 genes could be transferred between A. baumannii via the plasmid group GR6 or class 1 integrons (Int1) (Leungtongkam et al., 2018b). We were unable to find Int1 as well as other classes (Int2 and Int3) in the genome of A. baumannii AB329, which was consistent with a study by Ploy et al. (2000).
Prophages are important MGEs that encode toxins, enzymes, or drug resistance genes that allow their host to become more virulent and contribute to the evolution of pathogenic bacteria. In silico analysis by Loh et al. (2020) identified numerous ARGs that encoded beta-lactamase enzymes, N-acetyltransferases, aminoglycoside phosphotransferases, and a macrolide efflux pump in 177 prophages identified in A. baumannii genomes. However, we detected only the gene encoding the MFS transporter in the genome of A. baumannii AB329. The presence of the MFS transporter was reported in the prophage of A. baumannii NCIMB8209, which is involved in DNA transport and is necessary for biofilm formation (Repizo et al., 2020).
Accessory genes derived from HGT are found in typical regions known as GIs. Previous studies identified 63 GI loci in A. baumannii, and genes located within G4aby, G4abn, and G5abn were found to correspond to the resistance regions previously described as AbaR1, AbaR3, and AbaR4 (Di Nocera et al., 2011). AbaR4 was found in the genome of A. baumannii AB329. The AbaR4-type resistance island was the predominant type revealed to be a clone prevalent in most Asian countries; however, diverse variants of ARGs located within the island were found (Kim, Park & Ko, 2012;Kim et al., 2013). IS is a short DNA sequence that plays an extensive role in bacterial adaptation to antibiotic selective pressures. A previous study on 976 A. baumannii genomes detected 29 IS elements (Wright et al., 2017). ISAba1 is widely distributed in A. baumannii and plays a major role in the transfer and expression of bla OXA-23 and bla ADC (Turton et al., 2006;Mugnier, Poirel & Nordmann, 2009;Joshi et al., 2017). In this study, 17 ISAba1 genes were detected in A. baumannii AB329 and were found upstream/downstream of bla ADC (formaly bla ampC ) and bla OXA−133. A previous report stated that the bla NDM−1 gene was located within transposon Tn125 and was bracketed by two copies of ISAba125. In this study, the bla NDM−1 gene was found to be absent in A. baumannii AB329; instead, ISAba125 was observed to be located upstream and downstream of aph(3 )-VIa in pAB329b.

CONCLUSIONS
In this study, we presented a whole-genome analysis of A. baumannii AB329, an XDRAB strain isolated from Thailand. The A. baumannii AB329 genome contained MGEs, such as two plasmids, one intact prophage, 34 IS elements, and 19 GIs. Most ARGs were located in MGEs, suggesting that these MGEs function as major mechanisms for the dissemination of ARGs in A. baumannii.
• Rapee Thummeepak performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.
• Udomluk Leungtongkam performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.
• Kannipa Tasanapak conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.
• Aunchalee Thanwisai conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.
• Sutthirat Sitthisak conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The protocol was approved by Naresuan University Institutional Biosafety Committee.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The complete genome is available at NCBI GenBank: CP091452 (chromosome), CP091453 (pAB329a), and CP091454 (pAB329b).